A crucial event in the development of NK cell-mediated cytotoxicity is the receptor-stimulated secretion of granule-derived proteins. Prior to NK cell activation, pre-formed granules containing an array of cytotoxic and proteolytic molecules gather beneath the cell surface. With NK cell receptor recognition of susceptible targets, intracellular biochemical signals are generated that direct the final transport of the granules to the inner surface of the plasma membrane and the subsequent exocytotic fusion events. The long-term goals of this project are to define the relationship between specific signal transduction pathways and the regulation of granule exocytosis. For this proposal, experimental systems have been developed in which intact or permeabilized preparations of cloned NK cells can be used for the direct manipulation and evaluation of the relevant pathways. Preliminary work suggests that the transmembrane signaling events involve not only the selective activation of specific phospholipase C (PLC) isoforms, but other second messengers (e.g. protein tyrosine kinases, G proteins) also critically regulate the secretory response. The hypotheses formulated from these preliminary observations are tested in the following specific aims: 1) Characterize PLC-gamma activation in NK cells and its role in receptor-stimulated granule exocytosis; 2) Evaluate protein tyrosine-kinase-dependent, but PLC-independent transmembrane signals that also regulate NK cell secretion; 3) Analyze the role of G protein-mediated signals in the downstream propagation of signals leading to exocytosis; and 4) Evaluate the effects of IL-2 and the proto-oncogene p56(lck) on the secretory response. Together these studies will provide an experimental basis for understanding the molecular events that are involved in the regulation of NK cell secretion, and will, in a broader context, advance our understanding of fundamental processes involved in transmembrane signaling and lymphocyte activation.